Exploring ferredoxin-dependent glutamate synthase as an enzymat- ic

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Exploring ferredoxin-dependent glutamate synthase as an enzy-matic bioelectrocatalyst Fei Wu, Ping Yu, Xiaoti Yang, Zhongjie Han, Ming Wang, and Lanqun Mao J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.8b08020 • Publication Date (Web): 23 Sep 2018 Downloaded from http://pubs.acs.org on September 23, 2018

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Journal of the American Chemical Society

Exploring ferredoxin-dependent glutamate synthase as an enzymatic bioelectrocatalyst Fei Wu,†,‡ Ping Yu,†,‡ Xiaoti Yang,†,‡ Zhongjie Han,† Ming Wang,†,‡ and Lanqun Mao†,‡,* †

Beijing National Laboratory for Molecular Science, Key Laboratory of Analytical Chemistry for Living Biosystems, Institute of Chemistry, the Chinese Academy of Sciences (CAS), Beijing 100190, China ‡

University of CAS, Beijing 100049, China. CAS Research/Education Center for Excellence in Molecule Science, Beijing 100190, China.

Supporting Information Placeholder ABSTRACT: Ferredoxin-dependent glutamate synthase (FdGltS) is reported as an enzymatic bioelectrocatalyst for the first time. By configuring mediated electrochemical interfaces with mediators of different redox potentials, we realize bioelectrosynthesis or bioelectrooxidation of glutamate with recombinant Fd-GltS from cyanobacteria. Particularly, bioelectrocatalytic oxidation of glutamate by Fd-GltS is demonstrated to be oxygen independent. This study reinforces a new catalytic option for developing enzymatic bioelectronic devices for powering, sensing or synthesis.

Enzymatic bioelectrocatalysis basically investigates or utilizes heterogeneous electron transfer between the electrode and redox enzymes by electric currents. It has been making important progress over a long history for both fundamental and practical interests, in either unraveling redox control of mass and energy flow in cell metabolism in the language of enzyme kinetics or developing bioelectronic de1 vices for energy conversion, biosensing and electrosynthesis. Nature undoubtedly provides a broad diversity of enzyme candidates to meet bioelectrocatalytic demands, ranging 2 3 from extensively employed oxidases and dehydrogenases to 4 5-10 fragile hydrogenases, nitrogenases and complex multicen11-13 tered entities like photosystems and electron transport 14,15 chain. However, the mysterious world of metabolic redox proteins/enzymes remains largely unexplored while emerging with attractive properties and functions. Here, we introduce a new enzymatic bioelectrocatalyst named ferredoxindependent glutamate synthase (Fd-GltS) that exhibits unique electrochemical behavior. Fd-GltS is a key component in ammonia assimilation during nitrogen fixation, solely found participating in photoconversion and amino acid metabolism in higher green plants and microorganisms. It is a multicentered redox enzyme that catalyzes reductive biosynthesis of L-glutamate (or L-glutaric acid, L-GLU) from L-glutamine (L-GLN) and 2-oxoglutarate (2-OXG), using Fd (a small iron-sulfur protein) as a coen16,17 zyme. Up till now, only the crystallographic structure of

18

Fd-GltS from cyanobacteria has been resolved. Limited re18-24 searches on Fd-GltS has been focused on the structural, 25-27 28,29 redox, and functional properties, whilst its electrocatalytic aspect has never been studied. This paper provides the first exploration of the cyanobacterial Fd-GltS in catalyzing either bioelectrosynthesis of L-GLU or bioelectrochemical oxidation of L-GLU at a mediated electrochemical interface. Fd-GltS from cyanobacterium Synechocystis sp. PCC 6803 is a monomeric iron-sulfur flavoprotein of 160~180 kDa. As Figure 1 shows, it has two structurally and functionally distinct major domains, an amidotransferase domain (top part) and a Flavin mononucleotide (FMN) and iron-sulfur binding domain (bottom part). In the “redox” domain, a [3Fe4S] cluster is located near the protein surface to readily accept electrons. FMN is buried a little deeper, 0.8 nm away from the [3Fe4S] cluster. Besides, there are two more supplementary domains, a central domain linking two catalytic domains and a C-terminal domain that may help the synchronization of 18,19 distant active sites.

Figure 1. Crystallographic structures of Fd (PDB: 1OFE) and Fd-GltS (PDB: 1OFD) from Synechocystis sp. PCC 6803. Purple, yellow and red spheres respectively represent the amidotransferase site, FMN, [3Fe4S] cluster in Fd-GltS and [2Fe2S] cluster in Fd.

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interface where diffusional MV shuttles electrons between a bare

Figure 2. Schematic illustration (A) and cyclic voltammograms of methyl viologen-mediated bioelectrosynthesis of L2+ GLU in 50 mM Tris-HCl (0.1 mM MV , 7 µM Fd, 5 mM 2OXG, 150 mM NaCl, pH 8.0) in the absence (B) and presence (C) of Fd-GltS (2.25 µM) under anaerobic condition (scan -1 rate, 10 mV s ). Recombinant Fd and Fd-GltS (cyanobacterium Synechocystis sp. PCC 6803) were overexpressed in E. coli and purified to homogeneity, confirmed by respective intense protein bands of ca. 12 kDa and 170 kDa on SDS-PAGE gel (Figure S1). UV-Vis spectrum of the purified Fd displays characteristic absorption peaks of [2Fe2S] cluster at 425 and 460 nm, and that of the purified Fd-GltS shows absorption peaks of FMN and [3Fe4S] cluster at 345, 417, 440 and 476 nm (Figure S2), proving that our recombinant Fd and Fd-GltS are intact holoproteins. Biocatalytic synthesis of L-GLU by Fd-GltS is powered by naturally existing reducing equivalents that deliver electrons to Fd-GltS through the labile [2Fe2S] cluster of Fd. Simultaneous binding of 2-OXG to the FMN site and reduced Fd to the region of [3Fe4S] cluster in Fd-GltS triggers protein conformational change and then the distant activation of the amidotransferase site, where the N-terminal cysteine (Cys-1) hydrolyzes L-GLN to one molecule of L-GLU and one molecule of NH3. Ammonia is quickly transported to 2-OXG by an internal substrate channel (2.4 nm) to form 2-iminoglutarate that is reduced by intramolecularly transferred electrons from reduced Fd to finally form a second molecule of L17,19 GLU. In the present work, we used a sodium dithionite (DT)2+ methyl viologen (MV ) system as the artificial reducing equivalents. DT acted as the primary electron donor and 2+ ●+ reduced colorless MV to the purple cation radical (MV ) with a strong absorption at 604 nm (A604). Such highly re2+ ductive radicals can be quickly oxidized back to MV by Fd, yielding reduced Fd to initiate the biocatalytic scheme. We observed a continuous decrease of A604 when Fd, Fd-GltS, 22+ OXG and L-GLN were added into a DT-reduced MV solu●+ tion (Figure S3). Consumption of MV demonstrated the synthase activity of GltS. Heterogeneously, molecular electron donors (i.e., DT) can be replaced by electrodes to perform bioelectrosynthesis of L-GLU. Figure 2A depicts a mediated bioelectrocatalytic

Figure 3. Schematic illustration (A) and cyclic voltammograms of bioelectrooxidation of L-GLU mediated by PMS (B), DCIP (C) or ferricyanide (D) in 10 mM PBS (0.1 mM ferricyanide, pH 7.4, 600 nM GltS) under aerobic condition (scan -1 rate, 10 mV s ). glassy carbon electrode and solubilized Fd and Fd-GltS. Cy2+ clic voltammogram (CV) of MV in Figure S4 shows that it ●+ can be electrochemically reduced to MV at ca. -0.65 V (vs. Ag/AgCl). Further reduction at more negative potential (0.99 V) generates inactive neutral MV. Therefore, bioelectrocatalytic investigation was conducted in a narrower potential window. As can be seen in Figure 2C, adding 1 mM LGLN into the bulk solution containing Fd, Fd-GltS and 2-2 OXG produced a reductive turnover current of ca. 1 µA cm at -0.65 V. Meanwhile, oxidative peak current at -0.56 V de-2 creased by 0.66 µA cm . In comparison, adding substrate 2+ caused no change to non-turnover currents of MV in the absence of Fd-GltS (Figure 2B). Notably, we observed no catalytic current in the absence of Fd (data not shown). In ●+ other words, MV is not efficient in delivering electrons directly to Fd-GltS (consistent with previously reported assay 20 results). Nevertheless, the Fd/Fd-GltS complex is capable of catalyzing bioelectrosynthesis of L-GLU through a mediated electron transfer pathway. Besides glutamate synthesis, glutamate oxidation can also be catalyzed by Fd-GltS. As reported, Fd-GltS behaved as a 19 glutamate dehydrogenase in no need of Fd. To test its activity of catalyzing the back reaction, we choose iodonitro29 tetrazolium chloride (INT) as the ultimate acceptor of electrons from L-GLU. INT is a commonly used colorimetric indicator of dehydrogenases, absorbing strongly at 490 nm (A490) once reduced. A cocktail of INT, L-GLU and Fd-GltS displayed an increasing A490 (Figure S5), indicating dehydrogenase-like activity of the recombinant Fd-GltS. Having proved that L-GLU can reduce FMN in Fd-GltS, we established a much simpler mediated interface on bare glassy carbon electrode as illustrated in Figure 3A. In this proposed scheme, artificial mediators with higher redox potential allows direct electron transfer from FMN (mid-point


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potential is -180 mV vs. NHE ). In this regard, we examined three different mediators, phenozine methosulfate (PMS), 2,6-dichlorophenolindophenol (DCIP) and ferricyanide. Control CVs

Figure 4. Plot of oxidative current responses (solid spheres) to serial titrations of L-GLU fitted to a Michaelis-Menten curve (solid line). Bulk solution: 10 mM PBS (0.2 mM ferricyanide, 720 nM Fd-GltS, pH 7.4). Potential: +0.40 V vs. Ag/AgCl for ferricyanide and +0.1 V vs. Ag/AgCl for DCIP. without enzyme in Figure S6 determined their respective E1/2 values under test condition to be -144 mV, -2 mV and +223 mV (vs. Ag/AgCl). Figure 3B-D lists corresponding bioelectrocatalytic results with Fd-GltS. At PMS-mediated interface, no catalytic current was generated upon introduction of LGLU, while CV shifted to the anodic direction when L-GLU was present with the enzymes and DCIP. The backgroundsubtracted reductive current at -0.027 V decreased by 0.56 -2 µA cm , but the oxidative current at +0.017 V increased by -2 only 0.22 µA cm . Larger catalytic current was obtained at higher overpotential, such as an oxidative current increase of -2 1 µA cm at +0.10 V. Ferricyanide-mediated bioelectrocatalysis gave the similar trend that turnover current of glutamate oxidation became significant at more positive potentials. In Figure 3D, addition of 1 mM L-GLU produced an oxidative turnover current (non-catalytic current subtracted) of 0.20 -2 -2 µA cm at +0.30 V and 2.28 µA cm at +0.38 V, with the re-2 ductive current at 0.13 V decreased by 3.11 µA cm . It is clear to see that the catalytic current density goes larger as E1/2 of the mediator shifts positively, which can be attributed to enhanced driving force for the electron flow from the higher energy level in FMN to the lower energy level in mediators. Larger oxidation overpotential is required to overcome the kinetic and diffusional barrier in coupling chemical reaction at the active site of free Fd-GltS and electrooxiation of ferrocyanide at the electrode surface. Bioelectrocatalytic kinetics of Fd-GltS for glutamate oxidation was investigated by measuring current response to substrate titration in amperometric mode. Concentrations of L-GLU from 100 nM to 20 mM were studied, and steady-state current was monitored at 0.1 V (DCIP) or 0.4 V (ferricyanide). Figure S7 has shown the staircase current increases with [LGLU], and that the lowest [L-GLU] inducing an obvious current elevation with DCIP (1 µM) and with ferricyanide (2 µM) are comparable. Oxidative current density was plotted vs. [LGLU] in Figure 4. In the low substrate concentration regime (1 µM ~ 20 µM) a linear relationship between rapid current

increase and [L-GLU] can be extracted (Figure S7C and D). Then GltS became saturated by the substrate at concentrations higher than 5 mM. Using a Michaelis-Menten approximation, we determined kinetic parameters of Fd-GltS toward L-GLU oxidation. There is no statistical difference in Km values of DCIP- (0.62 ± 0.05 mM) or ferricyanide-mediated bioelectrocatalysis (0.66 ± 0.12 mM), which are comparable with reported value (0.33 ± 0.1 mM) determined by homoge29 neous assays , but ferricyanide is proved as a better mediator with a larger maximum current density (3.48 ± 0.42 µA -2 -2 cm ) as compared to DCIP (2.67 ± 0.25 µA cm ). Although we assumed that [3Fe4S] cluster was not involved during glutamate oxidation, it can be reduced by glu19 tamate as suggested by Mattevi et al. This seemed to be supported by studies on redox properties of Fd-GltS by Knaff 26 et al. They measured the mid-point potentials of the two redox centers that appeared close to each other (-180 mV vs. NHE for FMN and -225 mV vs. NHE for [3Fe4S] cluster), and electron transfer between FMN and the iron-sulfur site may be reversible. There is no doubt that FMN should be the pri28 mary electron acceptor during glutamate oxidation, but the question remains: does mediators like ferricyanide accept electrons from reduced [3Fe4S]? In this case, oxygen will be an inevitable interference, because it may easily take elec29 trons from the labile reduced iron-sulfur site. In order to test whether our hypothetical interfacial scheme was real, we studied the oxygen dependency of Fd-GltS-catalyzed glutamate oxidation. Non-catalytic and catalytic CVs were collected with GltS and ferricyanide under N2-saturated and airsaturated conditions, and no differences were witnessed as dissolved [O2] changed (Figure S8). Moreover, we compared current response to N2 bubbling under non-catalytic (no LGLU added) and catalytic (1 mM L-GLU) conditions. In Figure S9, small current increase was caused by injection of N2 without L-GLU, probably due to the stirring effect by gas bubbling and protection of reduced ferricyanide (or ferrocyanide) from oxygen, and such current change was almost the same even when catalysis was occurring. Taken all together, oxygen did not interfere with the communication between ferricyanide and Fd-GltS, thus excluding the possibility of participation of [3Fe4S] during the heterogeneous electron transfer. In conclusion, we have described the electrochemical exploration of Fd-GltS as a new member introduced to the family of bioelectrocatalysts. The unique capability of catalyzing either forward synthesis or backward oxidation by choosing different redox mediators makes it an intriguing enzyme to attract a broad interest in the fields of bioelectrochemistry and bioenergetics. In particular, current bioelectronic devices utilizing oxidases or dehydrogenases for energy harvest and conversion from abundantly existed biomolecules, such as glucose and amino acids, frequently share issues of oxygen dependency, hydrogen peroxide interference, mediator instability or large overpotential. An important implication here is that Fd-GltS can help developing new-generation glutamate-based biofuel cells and biosensors for two reasons, one being oxygen independency and the other being direct electron transfer with the electrode as its redox centers are close to enzyme surface. Hence, a future direction in studying Fd-GltS (also undergoing in our lab) is building an efficient direct contact between Fd-GltS and conducting surfaces by a variety of enzyme immobilization strategies. With


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this approach, we anticipate an in-depth investigation of bioelectrocatalytic mechanisms of Fd-GltS (substrate channeling and self-regulation) and generation of glutamate biofuel cells or biosensors for broad applications.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Experimental details and supplemental data including enzyme characterization and electrochemical measurements.

AUTHOR INFORMATION Corresponding Author [email protected]

Notes The authors declare no competing financial interests.

ACKNOWLEDGMENT The authors are grateful for financial support from the National Science Foundation of China ((Grant Nos. 21790390, 21790391, 21435007, 21621062 for L.M., and 21874139 for F. W.), the Strategic Priority Research Program of Chinese Academy of Sciences (Grant No. XDB30000000), and the Chinese Academy of Sciences (QYZDJ-SSW-SLH030).

(17) van den Heuvel, R. H. H.; Curti, B.; Vanoni, M. A.; Mattevi, A. Cell. Mol. Life Sci. 2004, 61, 669-681. (18) van den Heuvel, R. H. H.; Svergun, D. I.; Petoukhov, M. V.; Coda, A.; Curti, B.; Ravasio, S.; Vanoni, M. A.; Mattevi, A. J. Mol. Biol. 2003, 330, 113-128. (19) van den Heuvel, R. H. H.; Ferrari, D.; Bossi, R. T.; Ravasio, S.; Curti, B.; Vanoni, M. A.; Florencio, F. J.; Mattevi, A. J. Biol. Chem. 2002, 277, 24579-24583. (20) Navarro, F.; Martin-Figueroa, E.; Candau, P.; Florencio, F. J. Arch. Biochem. Biophys. 2000, 379, 267-276. (21) Suzuki, A.; Rothstein, S. Eur. J. Biochem. 1997, 243, 708-718. (22) Pelanda, R.; Vanoni, M. A.; Perego, M.; Piubelli, L.; Galizzi, A.; Curti, B.; Zanetti, G. J. Biol. Chem. 1993, 268, 3099-3106. (23) Marques, S.; Florencio, F. J.; Candau, P. Eur. J. Biochem. 1992, 206, 69-77. (24) Hirasawa, M.; Solis, J.; Vaidyanathan, N.; Srivastava, A. P.; Wynn, R. M.; Sutton, R. B.; Knaff, D. B. Photosynth. Res. 2017, 134, 317-328. (25) Hirasawa, M.; Hurley, J. K.; Salamon, Z.; Tollin, G.; Markley, J. L.; Cheng, H.; Xia, B.; Knaff, D. B. Biochim. Biophys. Acta 1998, 1363, 134-146. (26) Hirasawa, M.; Hurley, J. K.; Salamon, Z.; Tollin, G.; Knaff, D. B. Arch. Biochem. Biophys. 1996, 330, 209-215. (27) Hirasawa, M.; Robertson, D. E.; Ameyibor, E.; Johnson, M. K.; Knaff, D. B. Biochim. Biophys. Acta 1992, 1100, 105-108. (28) Vanoni, M. A.; Dossena, L.; van den Heuvel, R. H. H.; Curti, B. Photosynth. Res. 2005, 83, 219-238. (29) Ravasio, S.; Dossena, L.; Martin-Figueroa, E.; Florencio, F. J.; Mattevi, A.; Morandi, P.; Curti, B.; Vanoni, M. A. Biochemistry 2002, 41, 8120-8133.

REFERENCES (1) Cracknell, J. A.; Vincent, K. A.; Armstrong, F. A. Chem. Rev. 2008, 108, 2439-2461. (2) Wilson, R.; Turner, A. P. F. Biosens. Bioelectron. 1992, 7, 165185. (3) Lobo, M. J.; Miranda, A. J.; Tunon, P. Electroanalysis 1997, 9, 191-202. (4) Hambourger, M.; Gervaldo, M.; Svedruzic, D.; King, P. W.; Gust, D.; Ghirardi, M.; Moore, A. L.; Moore, T. A. J. Am. Chem. Soc. 2008, 130, 2015-2022. (5) Milton, R. D.; Abdellaoui, S.; Khadka, N.; Dean, D. R.; Leech, D.; Seefeldt, L. C.; Minteer, S. D. Energy Environ. Sci. 2016, 9, 25502554. (6) Hu, B.; Harris, D. F.; Dean, D. R.; Liu, T. L.; Yang, Z.-Y.; Seefeldt, L. C. Bioelectrochemistry 2018, 120, 104-109. (7) Fourmond, V.; Leger, C. Angew. Chem. Int. Ed. 2017, 56, 43884390. (8) Cai, R.; Milton, R. D.; Abdellaoui, S.; Park, T.; Patel, J.; Alkotaini, B.; Minteer, S. D. J. Am. Chem. Soc. 2018, 140, 5041-5044. (9) Milton, R. D.; Cai, R.; Abdellaoui, S.; Leech, D.; De Lacey, A. L.; Pita, M.; Minteer, S. D. Angew. Chem. Int. Ed. 2017, 56, 2680-2683. (10) Milton, R. D.; Cai, R.; Sahin, S.; Abdellaoui, S.; Alkotaini, B.; Leech, D.; Minteer, S. D. J. Am. Chem. Soc. 2017, 139, 9044-9052. (11) Yehezkeli, O.; Tel-Vered, R.; Wasserman, J.; Trifonov, A.; Michaeli, D.; Nechushtai, R.; Willner, I. Nat. Commun. 2012, 3. (12) Kato, M.; Cardona, T.; Rutherford, A. W.; Reisner, E. J. Am. Chem. Soc. 2012, 134, 8332-8335. (13) Kato, M.; Zhang, J. Z.; Paul, N.; Reisner, E. Chem. Soc. Rev. 2014, 43, 6485-6497. (14) Takamura, K.; Mori, A.; Kusu, F. Bioelectrochem. Bioenerg. 1982, 9, 499-508. (15) Pelster, L. N.; Minteer, S. D. Acs Catal. 2016, 6, 4995-4999. (16) Vanoni, M. A.; Curti, B. Cell. Mol. Life Sci. 1999, 55, 617-638.


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TOC graphic:



Figure 1 65x76mm (240 x 240 DPI)


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Figure 2 128x99mm (240 x 240 DPI)



Figure 3 134x120mm (240 x 240 DPI)


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Figure 4 259x209mm (72 x 72 DPI)


Exploring ferredoxin-dependent glutamate synthase as an enzymat- ic

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